We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (sigma). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topo-isomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with sigma < -0.05 is very significant because these temperatures for early melting are below physiological temperatures.